Apparatus for use in liquid sample analysis

ABSTRACT

An electrophoretic cell having an elongate electrolytic expanse is enveloped between an overlayer and a substrate, the overlayer including a closable opening through which a liquid sample may be applied to a preselected location on the elongate electrolytic expanse. The cell provides for separation of the constituents of the sample on application or preselected electric voltage to the cell electrodes.

F. W. GODSEY, JR

APPARATUS FOR USE IN LIQUID SAMPLE ANALYSIS Filed June 26. 1972 FIG.

,10 ,12 I4a\ lflflhlll [I43 WI IHHM! i 4 will 14c 5W Noam Ib III 4 ll HIII! L mb "W 206-1 '1 United States Patent 3,826,734 APPARATUS FOR USE INLIQUID SAMPLE ANALYSIS Frank W. Godsey, In, St. Petersburg, Fla.,assignor to Bio-Medical Sciences, Inc., Fairfield, NJ. Filed June 26,1972, Ser. No. 266,449 Int. Cl. 301k /00 U.S. Cl. 204-499 ClaimsABSTRACT OF THE DISCLOSURE An electrophoretic cell having an elongateelectrolytic expanse is enveloped between an overlayer and a substrate,the overlayer including a closable opening through which a liquid samplemay be applied to a preselected location on the elongate electrolyticexpanse. The cell provides for separation of the constituents of thesample on application or preselected electric voltage to the cellelectrodes.

FIELD OF THE INVENTION 1 This invention relates to apparatus for use inthe analysis of fluid samples and more particularly to test apparatusfor use in determining protein fractions in human body fluids such asblood.

BACKGROUND OF THE INVENTION Various procedures are presently known forseparating the protein fractions of blood serum for quantificationthereof, e.g., salting-out of the fractions, precipitation of thefractions using predetermined concentrations of organic solvents,isoelectric, cationic and anionic precipitation, and the use ofcentrifuges and electrophoresis. As presently implemented, all of thesemethods are practiced with laboratory discipline and involve thecollection of a sample of whole blood in the order of cubic centimeters,transportation of the collected sample to a laboratory facility,separation of blood serum from the whole blood sample and finally, thequalitative and quantitative analysis of the serum. In the most widelypracticed of the foregoing procedures, namely, that involvingelectrophoresis, the whole blood sample together with a clotting agentis placed in a centrifuge and the resulting blood serum is applied to amedium many inches in length and pre-wetted with an electrolyte of pHlevel sufficient to enable the medium to support the migration ofprotein fractions. The medium is disposed across a trough-likeelectrolyte container with the opposed medium end portions immersed incontainer electrolyte. An electric voltage of one hundred or more voltsis applied between a pair of electrodes supported by the container andelectrically connected by container and medium electrolyte. Anelectrical field is thus applied to the serum and induces migration ofthe protein fractions along the medium, fractions involving smallermolecules of higher electrical charge moving at a faster rate than otherfractions involving larger molecules of lesser electrical charge. Withthe passage of time, the participating protein fractions of differenttype migrate to distinct zones having definite boundaries. Uponcompletion of a preselected time, the applied voltage is discontinuedand the medium is subjected to treatment which fixes the proteinfractions in their respective migra tion zones. Quantification of theseparated and fixed fractions is attained by chromatography, i.e., bystaining the medium and comparing color therein with a standard, or bypassing the medium through a densitometer.

As is evident from the foregoing, the presently known procedures forblood serum analysis require apparatus not ordinarily available for useoutside a laboratory and demand the discipline of a skilled technician.In this re- 3,826,734 Patented July 30, 1974 SUMMARY OF THE INVENTION Itis an object of the present invention to provide improved apparatus foruse in the analysis of fluid samples.

It is a more particular object of the invention to provide improvedelectrophoretical apparatus for use in analyzing blood serum.

In the efficient attainment of these and other objects, the presentinvention provides elec'trophoretical apparatus usable in non-laboratoryenvironment for the receipt of a fluid sample and for separatingfractions thereof into distinct zones for quantification. In itsparticularly preferred form, such apparatus includes, on a commonsubstrate, a blood serum protein fraction-discriminating cell disposedin a sealed container openable at the time of ap paratus use and afurther cell adapted to supply all operating power required by theprotein fraction discriminating cell.

The foregoing and other objects and features of the invention will beevident from the following detailed description of preferred embodimentsthereof and from the drawings wherein like parts are identified by likereference numerals throughout.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view of oneembodiment of apparatus in accordance with the invention.

FIG. 2 is a front elevational view of the FIG. 1 apparatus taken insection along the plane II-II of FIG. 1.

FIG. 3 is a partial view taken in section of an alternate form of theFIG. 1 apparatus.

FIG. 4 is a plan view of a second embodiment of apparatus in accordancewith the invention.

FIG. 5 is a plan view of a third embodiment of apparatus in accordancewith the invention.

FIG. 6 is a side elevational view of the FIG. 5 apparatus taken insection along the plane VI-VI of FIG. 5.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Test apparatus 10 ofFIGS. 1 and 2 includes a substrate 12 to which is secured the marginalportions 14a-d of a film 14. The substrate and film are comprised ofchemically inert, electrically on-conductive materials. Film 14 issubstantially transparent to light and the substrate may also have thischaracteristic, Film 14 includes an opening 14:: extending therethroughfor purposes discussed below. This opening is closed by removablesealing strip 16 preferably equipped with a tab 16a to facilitateremoval thereof. First and second electrodes 18 and 20 are spacedlydisposed on substrate 12 interiorly of film 14 and are separated byelongate means in the form of a strip member 22. Electrodes 18 and 20are preferably comprised of like or dissimilar metals and strip member22 is typically a strip of paper, cellulose acetate or polyethylenefoam, opposed end surfaces thereof preferably abutting the facingsurfaces of electrodes 18 and 20, respectively. In assembling the FIG. 1apparatus, electrodes 18 and 20 are preferably formed on substrate 14,e.g., by vacuum deposition or spraying techniques, and strip member 22is treated to provide electrolytic connection of the electrodes of pHlevel supporting migration of the constituents of a liquid sample understudy, e.g., the protein fractions of blood serum, upon application ofpreselected electrical voltage to the electrodes. In the case of bloodserum, the

electrolyte may comprise sodium diethylbarbiturate buffer, knowncommercially by the trademark Veronal and having a pH of 8.6. Suchtreatment of the strip member may comprise pre-wetting the stripthroughout or applying electrolyte to surface 22a thereof to provide afilm of electrolyte thereon, constituent migration desirably occurringin such electrolyte film and not within the strip member. Film 14 may befabricated so as to provide nominal clearance between itself and surface22a. Since the film is secured to the substrate along its marginalportions, the film comprises a containment layer for the electrophoreticcell comprised of electrodes 18 and 20 and the elongate meanstherebetween. By virtue of the seals between members 12 and 14 andbetween 14 and 16, loss of electrolyte is minimized and apparatus shelflife is enhanced.

Various alternative modes of assembling the apparatus of FIGS. 1 and 2may be employed. For example, the assembly of elements may beaccomplished with strip member 22 in dry state, electrolyte being addedprior to securement of sealing strip 16 to film 14 or at the time of useof the apparatus.

Conductors 18a and 20a extend from electrodes 18 and 20, respectively,through film 14 to provide for the connection of a voltage source 24 tothe electrodes.

In use of the apparatus of FIGS. 1 and 2, sealing strip 16 is peeledaway from film 14 at the time of use and pre-measured quantity of bloodserum, less than a drop as contrasted with above-discussed cubiccentimeters laboratory quantity, is applied to opening 14c of the cellcontainment layer. After a short time period, the residue of the drop iswiped away from opening 14c. At this time, conductors 18a and 20a areconnected to voltage source 24, which provides a voltage of magnitudesufficient to provide the field intensity (volts/cm.) throughout theelectrolyte film on strip member 22 required for protein fractionseparation.

The elongate means supporting protein fraction separation in theapparatus of FIGS. 1 and 2 may comprise a strip member of severalcentimeters in length, as contrasted with the separation supportingmeans in prior laboratory-type apparatus of many inches in length.Accordingly, to attain sufficient field intensity, e.g., 20 volts percentimeter, apparatus of FIGS. 1 and 2 requires a voltage sourceproviding tens of volts rather than the hundreds of volts required inprior laboratory-type apparatus. Strip member width is likewise reducedfrom that of the prior laboratory apparatus. The apparatus of FIGS. 1and 2 provides for the clear separation of protein fractions inshortened migration distances and in consequently shortened timeperiods. Such separation occurs on completion of a time periodpredeterminable in accordance with the distance between electrodes 18and 22, the voltage provided by source 24, the electrolyte employed, andlike factors influencing migration. Following separation, the elongatemeans is subjected to fixing, and may thereafter be stained andinspected by chromatography whereby each separated fraction isquantized.

Apart from such staining technique, the invention contemplates opticalreading of the separated constituent magnitudes. In this connection,substrate 12, film 14 and strip member 22 may all be comprised ofmaterial substantially transparent to light whereby light diffractionthrough the apparatus, attributable to protein fraction magnitude,provides a basis for quantizing. 'As discussed above, blood serumquantity employed in samples analyzed by the apparatus of the inventionis quite small. As-a result, a separation of the blood serum sample fromwhole blood .may be effected by the use of filter 3, microporous member26 may be disposed in the aperture 14e of film 14. Where whole blood isapplied to the member, clotting thereof occurs in the member and onlythe serum passes therethrough to strip member 22. Such microporousmember may incorporate a clotting agent to enhance the clotting of wholeblood therein.

means integral with the test apparatus. Referring to FIG.

Test apparatus 28 of FIG. 4 includes on its substrate 30 anelectrophoretic cell 32 constructed as in the case of theelectrophoretic cell of the apparatus of FIGS. 1 and 2. While in theapparatus of FIGS. 1 and 2, conductors 18a and 20a were connected to avoltage source 24, not integral with apparatus 10, correspondingconductors 18b and 20b of apparatus 28 are connectedto a galvanic cell34 disposed on substrate 30. The FIG. 4 apparatus is accordinglyself-contained as respects all elements needed in providing for theseparation of the constituents of a liquid sample. In the particularapplication of the apparatus to blood serum analysis, galvanic cell 34is required tov apply to conductors 18b and 20b a voltage in the orderof tens of volts, as discussed above, and is thus preferably comprisedof a plurality of discrete cells connected in series circuit betweenconductors 18b and 20b each discrete cell providing a complement of suchtotal voltage. In the illustrated embodiment of apparatus 28, three suchcells are illustrated each being commonly structured. By way of example,cell 36 may include first and second electrodes 38 and 40, respectivelyfractions of milligrams of carbon and zinc spacedly disposed onsubstrate 30 with suitable electrolytic connection therebetween. Suchelectrolytic connection may be provided by the use of anelectrolyteabsorbent member 42 comprised of paper or the like. Theelectrolyte for activating cell 36 may be introduced into member 42 atthe time of use as is illustrated schematically by removable sealingmember 43.

Use of the Fig. 4 apparatus parallels that of the FIGS. 1 and 2apparatus discussed above, the application of electrical voltage toelectrophoretic cell 32 of FIG. 4 being accomplished by activatinggalvanic cell 34. In the galvanic cell form illustrated schematically,activation occurs on addition of electrolyte to all the individualunits, e.g., 36. Where the galvanic cell is provided beforehand withelectrolyte, means may be incorporated for providing selectiveconnection of the galvanic cell to the electrophoretic cell to controlapplication of voltage to the electrophoretic cell. 1

Referring to FIGS. 5 and 6, apparatus 44 comprises a substrate 46 havinga surface depression 461: covered by cell containment layer 48. Suchdepression defines a trough-like receptacle for an electrophoretic cellincluding electrodes 52 and 54 and elongate means comprising a body ofelectrolyte 50. Layer 48 includes therein an opening 48a incommunication with a channel 56 extending to depression 46a. Layeropening 48a is closed by removable sealing member 58 having tab 58a. Onremoval of member 58, the sample is applied to layer opening 48a andthence through channel 56 into the electrolyte in depression 46a. Uponapplication of voltage to electrode conductors 52a and 54a, separationof the sample constituents occurs in electrolyte 50 between theelectrodes.

In the apparatus of FIGS. 4 and 5, the dimensions of depression 46a areselected with a view toward minimizing mixing of the sample with theelectrolyte and toward avoiding any migration of the sampleconstituents'in the absence of applied voltage. The mobilities of sampleconstituents are increased from sample constituent mobilities in thepreviously-discussed embodiments of apparatus in accordance with theinvention in view of the increased migration-supporting electrolytearea. Accordingly, lessened time for sample analysis is required. Thegreater ratio of surface area to volume in the apparatus of FIGS. 4 and5 as compared to the previously-discussed appara tus serves effectivelyto reduce temperature rises from electrical sources. In addition, lessersample volume is required. Where substrate 46 and cell containment layer48 are comprised of materials substantially transparent to light, theaforementioned optical reading of sample constituents may be practicedupon completion of constituent migration.

It is to be appreciated that various changes may be made in theforegoing particularly disclosed preferred embodiments of apparatusaccording with the invention.

For example, any of the vast number of materials having theaforementioned characteristics may be used in respect of the apparatussubstrates, cell containment members, sealing strips, electrodes,electrolytes and the like. Immobilizing additives, such as gellingagents, for the electrolytes are Within the contemplation of theinvention. Accordingly, the particularly disclosed embodiments areintended in an illustrative and not in a limiting sense. The true spiritand scope of the invention is defined in the following claims.

What is claimed is:

1. A self-contained electrophoretic cell comprising a chemically inert,electrically non-conducting substrate member, a pair of electrodes fixedto said substrate member, a thin strip member disposed on said substratemember between and in electrical contact with said electrodes, saidstrip being operable to support an electrophoretic medium, and achemically inert electrically non-conducting transparent film overlyingsaid strip and said electrodes and having its marginal portions sealedto said substrate member, said film having an opening operable to permitintroduction of a sample to said strip and closure means operable toseal said opening before and after sample introduction.

2. A cell as defined in claim 1 wherein said strip member is wettedthroughout by an electrolyte prior to sealing said film.

3. A cell as defined in claim 1 including a pair of conductors inelectrical connection with said electrodes and extending to the exteriorof said cell.

4. A cell as defined in claim 3 wherein said conductors are adapted forelectrical connection with an outside voltage source.

5. A cell as defined in claim 3 including a self-contained voltagesource in electrical contact with said electrodes through saidconductors.

6. A cell as defined in claim 1 wherein said strip member comprisespaper, cellulose acetate or polyethylene.

7. A cell as defined in claim 1 wherein said substrate- UNITED STATESPATENTS 794,864 7/1905 Kamperdylc 136-952 795,325 7/1905 Winters136-1252 968,154 8/1910 Hite 136-127 X 3,432,414 3/1969 Rand 204- G3,582,490 6/1971 Zemel 204-180 G 3,594,263 7/1971 Dwyer et a1. 204-180 SX 3,635,808 1/1972 Elevitch 204-180 G 3,674,678 7/1972 Post, Jr. et al.204-299 3,691,054 9/1972 CaWlcy 204-299 JOHN H. MACK, Primary ExaminerA. C. PRESCOTT, Assistant Examiner US. Cl. X.R. 204-180 G, 180 S

